Highly-purified recombinant reverse transcriptase

ABSTRACT

A plasmid for expression of Moloney Murine Leukemia Virus-derived reverse transcriptase in E. coli cells deficient in the expression of indiginous RNAse activity, a method for purification of the recombinant enzyme, and a composition comprising a cloned and purified reverse transcriptase opimized for use in cDNA and nucleic acid amplification procedures.

This is a file-wrapper continuation of Ser. No. 08/443,781, filed May18, 1995, now abandoned, which is a continuation of Ser. No. 08/221,804,filed Apr. 1, 1994, now abandoned, to which this application claimspriority.

BACKGROUND OF THE INVENTION

Retroviruses are a group of viruses whose genetic material consists ofsingle-stranded RNA. Following adsorption and entry of the retroviralRNA into the host cell, the viral RNA serves as a template for thesynthesis of a complementary DNA strand. The DNA is then madedouble-stranded through the action of an enzyme having DNA polymeraseactivity; it is this double-stranded DNA which integrates into the hostgenome. The RNA-directed DNA polymerase activity responsible for thesynthesis of complementary DNA from the viral RNA template is commonlycalled reverse transcriptase.

Retroviruses are of particular interest because a number of retroviruseshave been implicated as the causative agents of various cancers, andother diseases. A retrovirus, human immunodeficiency virus, is thecausal agent of acquired immunodeficiency syndrome (AIDS). Additionally,the reverse transcriptase enzymes themselves have become importantreagents in molecular biology because of their ability to makecomplementary DNA from almost any RNA template. Thus, reversetranscriptase is commonly used to make nucleic acids for hybridizationprobes and to convert single-stranded RNA into a double-stranded DNA forsubsequent cloning and expression.

Recently, reverse transcriptases have been used as a component oftranscription-based amplification systems. These systems amplify RNA andDNA target sequences up to 1 trillion fold. See e.g., Burg et al., PCTPatent Application WO 89/01050 (1988); Gingeras et al., PCT PatentApplication WO 88/10315 (1988); Davey and Malek, European PatentApplication EPO 0329822 (1988); Gingeras et al., European PatentApplication EPO 0373960 (1989); Malek and Davey, PCT Patent ApplicationWO 91/02814 (1989); Kacian and Fultz, European Patent Application EPO0408295 A2 (1990). All of these references are hereby incorporated byreference into this disclosure.

Some of the transcription-based amplification methods are exceptionallyconvenient since the amplification reaction according to these methodsis isothermal. Thus, these systems are particularly suited for routineclinical laboratory use in diagnostic tests. Detection of pathogenscausing infectious diseases and gene sequences associated with cancersor genetic diseases are among the most important uses of such tests.Reverse transcriptases are also employed as an initial step in someprotocols when the polymerase chain reaction (PCR) is used to amplify anRNA target. See Malek et al., U.S. Pat. No. 5,130,238 (1992); andMocharla et al., Gene 99:271-275 (1990). In such "RT-PCR" procedures,the reverse transcriptase is used to make an initial complementary DNA(cDNA) copy of the RNA target, which is then amplified by successiverounds of DNA replication.

The retroviral reverse transcriptases have three enzymatic activities: aRNA-directed DNA polymerase activity, a DNA-directed DNA polymeraseactivity, and an RNAse H activity. See Verma, The Reverse Transcriptase,Biochim. Biophys. Acta 473:1-38 (1977). The latter activity specificallydegrades RNA contained in an RNA:DNA duplex. Degradation of the RNAstrand of RNA:DNA intermediates by RNAse H is an important component ofsome transcription-based amplification systems and is to bedistinguished from unwanted degradation due to contaminating nucleases,which interferes with amplification.

A disadvantage of the transcription-based amplification systems is theirsensitivity to even trace amounts of nucleases. Since a number ofimportant diseases may yield samples containing very few target nucleicacid molecules, the detection of small amounts of the target is oftencrucial for an accurate and timely diagnosis. Indeed, the value oftarget amplification methods is most important when the number of targetmolecules is low. At low input levels of the target nucleic acids,unwanted degradation of RNA targets or RNA or DNA reaction intermediatescan lead to amplification failures and consequent misdiagnosis.Ribonuclease contamination is also a problem in RT-PCR reactions, sinceloss of the RNA target can lead to amplification failure.

Ribonucleases are relatively ubiquitous, and, in particular, are foundin high concentrations in a variety of biological materials, includingpreparations of retroviruses and in cells commonly used to expressrecombinant proteins. Ribonucleases frequently contaminate reversetranscriptase preparations from a variety of sources and have beenreported to interfere with synthesis of cDNAs, preparation of probes,and other uses besides target amplification alone. Often, an RNaseinhibitor is included in the reaction to minimize the deleteriouseffects of this contamination. See e.g., Maniatis et al., MolecularCloning: A Laboratory Manual 8.11-8.13 (2d ed. Cold Spring HarborLaboratory Press 1989), hereby incorporated by reference herein.

However, a number of substances commonly used to inhibit or inactivateRNAses, including detergents, chaotropes, organics, metals, proteasesand metals are inappropriate for use in target amplification systemssince they will inhibit the enzymes used for amplification as well.RNAse-inhibiting proteins such as human placental RNAse inhibitor,Blackburn et al., J. Biol. Chem. 252:5904 (1977) or rat liver RNAseinhibitor, Gribnau et al., Arch. Biochem. Biophys. 130:48-52 (1969), maybe unstable, are expensive, and can contribute additional interferingsubstances such as nucleic acids and RNAses that are not inhibited bythe inhibitor.

In addition to nucleases, traces of other enzymes, nucleic acids, andcertain buffer salts may interfere with amplification reactions. Whilethese substances are merely undesirable for many uses of reversetranscriptase, because of the nature of the amplification reaction it iscritical that the enzyme preparation contain as low an amount of them aspossible.

Isolation and purification of reverse transcriptase from various sourceshave been reported. In cases where the enzyme is isolated directly fromvirus particles, cells, or tissues, the cost is too high for widespreadcommercial use in diagnostic tests. See e.g., Kacian et al., Biochim.Biophys. Acta 46:365-83 (1971); Yang et al., Biochem. Biophys. Res.Comm. 47:505-11 (1972); Gerard, et al., J. Virol. 15:785-97 (1975); Liuet al., Arch. Virol. 55 187-200 (1977); Kato et al., J. Virol. Methods9:325-39 (1984); Luke, et al. Biochemistry 29:1764-69 (1990); Le Griceet al., J. Virol. 65:7004-07 (1991). Additionally, these methods havenot assured removal of substances that are significant inhibitors orcontaminants that interfere with the use of reverse transcriptase fortarget amplification reactions. Another important consideration in theuse of reverse transcriptases for a variety of purposes is the RNase Hactivity associated with the enzyme. The amount of RNase H activity andthe way in which the RNase H activities work in coordination with theRNA- and DNA-dependent reverse transcriptase activities are importantfeatures affecting the utility of the enzyme for various purposesincluding transcription-based amplification systems. Too much or toolittle activity, the wrong kind of activity (such as non-specificRNases), or activities poorly coordinated with DNA synthesis can alllead to reduced performance in a particular application. Proper balanceof the synthetic and degradative activities must be maintained; this isnot only a function of the particular reverse transcriptase enzyme used,but also is dependent on the ability of the purification protocol toremove the RNA and/or DNA degrading activities.

The cloning and expression of reverse transcriptases in bacterial hostshas been previously reported. Attempts to clone and express reversetranscriptase from avian myeloblastosis virus (AMV-RT) did not lead toproduction of significant amounts of the purified enzyme. This isapparently due to the fact that the AMV-RT consists of two polypeptidechains, the α and β chains, which must form a dimeric structure andundergo specific post-translational modifications in order to producefully active enzyme. These same modifications do not occur when the geneis expressed in E. coli.

By contrast to the avian viral RTs, many reverse transcriptases derivedfrom mammalian viruses consist of only one polypeptide chain; efforts toclone and express these enzymes have been more successful. Inparticular, Goff et al., U.S. Pat. No. 4,943,531 (1990) and Kotewicz etal., U.S. Pat. No. 5,017,492 have described methods for the purificationof reverse transcriptase derived from Moloney Murine Leukemia Virus(MMLV-RT) and expressed in E. coli, which methods form the basis for themajority of commercial reverse transcriptase preparations.

Many commercial preparations of reverse transcriptase have been foundunsuitable for use in target amplification and for other purposes due tonuclease contamination. See Sambrook, supra, previously incorporated byreference herein; Ryskov et al., Mol. Biol. Rep. 8:213-16 (1982). Otherproblems with commercial preparations of MMLV-RT may be related to analtered coordination between the DNA synthesis and RNAse H activities ofthe purified enzyme, reduced ability to bind and initiate synthesis atprimer sites or to read through regions of tight secondary structure, oralternately may be due to DNase and other protein contamination. SeeAgronovsky, A. A., Anal. Biochem. 203:163-65 (1992). Additionally,commercial preparations made using the previously available methods forpurification show significant lot-to-lot variability.

Moreover, due in part to the lengthy and labor-intensive purificationsemployed, the expense of the reagents and equipment employed forscale-up and the low yields of enzyme, the cost of such enzymes isprohibitive for their widespread commercial application in targetamplification systems.

It is therefore an object of the present invention to provide animproved form of reverse transcriptase having the correct balance of DNAsynthetic activities and RNAse H digestive activities, thereby beingparticularly suited for use in nucleic acid amplification methods.

It is another object of the present invention to provide a convenientsource of reverse transcriptase containing low Levels of contaminants,such as undesired RNAses, that interfere with transcription-basedamplification reactions by cloning and expressing a gene coding for anMMLV-RT enzyme having these properties in an E. coli host.

It is yet another object of the present invention to reduce the RNAseactivity associated with the enzyme prior to and following purificationby cloning and expressing the MMLV-RT gene in a ribonuclease-deficientstrain of E. coli.

It is another object of the present invention to develop a simplepurification scheme for the isolation of the enzyme.

It is a further object of the present invention to provide methods forthe purification of the enzyme that achieve high levels of purity of RTat a low cost.

SUMMARY OF THE INVENTION

The present invention features an expression vector or plasmidcontaining a cloned version of the gene for MMLV-RT which, when used totransform a suitable host cell such as E. coli, leads to the expressionof the gene and the generation of a gene product having the DNA- andRNA-directed DNA polymerase activities and RNAse H activity associatedwith retroviral reverse transcriptases.

The present invention also features a plasmid containing a gene forMMLV-RT inserted into a host cell which has a reduced level ofribonuclease activity as compared to wild-type strains.

The present invention also includes methods for the purification of theresulting enzyme from the host cells, such methods comprising suitablegrowth media, fermentation conditions, harvesting and storage of thecells, cell lysis and chromatography.

The present invention also features the enzyme produced by theexpression vectors, host cells, and purification procedures of thepresent invention. The enzyme is highly-purified and suitable for use innucleic acid amplification and other genetic engineering procedures.

Finally, the present invention features the use of the enzyme producedby the methods described herein for the synthesis of complementary DNAfor a variety of purposes, notably in transcription-based amplificationand RT-PCR reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and 1B: Construction of plasmid pUC18N

FIG. 2: Oligonucleotides used to construct plasmid pUC18N.

FIG. 3: Alignments of the ribosome binding sites.

FIG. 4: Oligonucleotides used in the construction of improved RBSvectors to modify the ribosome binding site and spacer region.

FIG. 5: Construction of plasmid pUC18 MMLV Sst-Hind.

FIG. 6: Construction of plasmid pUC18 MMLV III Tailed.

FIG. 7: Oligonucleotides used to construct the 3' end of plasmid pUC18MMLV Tailed.

FIGS. 8A to 8F: Construction of plasmids pUC18N MMLV III Gly and pUC18NMMLV Gly Tet(-).

FIGS. 9A to 9E: Construction of plasmids pUC18N SD9D MMLV Gly and pUC18NSD9D MMLV Gly Tet(-).

FIG. 10: Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) gel photograph of P-11 and Sephacryl S-200 fractions ofpurified MMLV-RT.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein the following terms have the indicated meanings unlessexpressly indicated otherwise.

By "selectable marker gene" is meant a DNA fragment encoding a genewhich, when carried and expressed by a host cell, is capable ofconferring a growth advantage to that host cell as compared to cells notcontaining the selectable marker gene when both are grown in a culturemedia of a given composition. For example, the gene encoding β-lactamasewill confer resistance to amplicillin on host cells containing thisgene, whereas cells not containing the gene will be sensitive toampicillin; thus only cells expressing the gene for β-lactamase willgrow in media containing amplicillin. Similarly, cells unable tocatabolize an essential amino acid will not grow in media not containingthat amino acid, whereas cells containing a gene allowing the cell tomake the essential amino acid will grow in the same media.

A selectable marker gene may be covalently linked, for example in aplasmid or expression vector, to one or more other gene or geneticelement as a means of identifying cells containing both the selectablegene and the "silent" gene(s) and/or genetic element(s).

By a "purified" nucleic acid or protein is meant a nucleic acid orprotein subjected to at least one step which removes cellular componentssuch as carbohydrates, lipids, unwanted nucleic acids, or unwantedproteins from the indicated nucleic acid or protein.

By "upstream" is meant to the 5' side of a given locus on a nucleic acidstrand, or in the case of a double stranded nucleic acid molecule, tothe 5' side of a particular locus with respect to the direction of genetranscription in that region of the nucleic acid molecule.

By "downstream" is meant to the 3' side of a given locus on a nucleicacid strand, or in the case of a double stranded nucleic acid molecule,to the 3' side of a particular locus with respect to the direction ofgene transcription in that region of the nucleic acid molecule.

By "T_(m) " is meant the temperature at which 50% of a population of adouble-stranded nucleic acid molecules, or nucleic acid molecules havinga double-stranded region, become single-stranded or thermally denatured.

By "recombinant" is meant that a nucleic acid molecule or protein is atleast partially the result of in vitro biochemical techniques. A"recombinant DNA molecule" is thus a non-naturally occurring molecule.Such recombinant molecules include, but are not limited to moleculeswhich comprise restriction endonuclease fragments, in vitro nucleic acidligation products, in vitro exonuclease fragments, and expressionvectors comprising heterologous genetic elements such as one or more ofthe following: promoters, repressor genes, selectable marker genes,temperature-sensitive DNA replication elements, structural genes, andthe like.

"Recombinant" proteins or enzymes are those not found in nature. Theseinclude purified protein preparations and proteins produced fromrecombinant DNA molecules. The latter proteins are usually expressed ina heterologous host cell, i.e., one not native to the protein or enzymein question. However, the gene encoding a recombinant protein may resideon an expression vector contained within a host cell of the same speciesas the organism from which the protein in question was derived.

By "truncated" is meant a smaller version of the gene or protein inquestion; with respect to the primary nucleotide or amino acid sequence,a truncated form of a reference nucleic acid or protein is one thatlacks one or more nucleotides or amino acids as compared to thereference molecule.

By "substantial sequence homology" is meant that a first nucleic acid orprotein molecule has a recognizably non-random similarity to a secondreference nucleic acid or protein over at least about 89% of itsnucleotide or amino acid sequence respectively.

By a nucleic acid or protein "domain" is meant at least one definiteregion of contiguous nucleotide or amino acid residues.

By "origin of replication" is meant a specific region of DNA at whichprimer production and initiation of DNA polymerase activity begins. Inthis specification, the term is used to mean a nucleic acid elementpresent on a DNA expression vector that allows the expression vector toincrease in copy number within a given host cell.

By "promoter" is meant a genetic element comprising a specific region ofDNA at which an RNA polymerase enzyme can bind and begin transcriptionof a DNA template, thus providing the first step of translating thegenetic information contained in the sequence of a nucleic acid into theproduction of a protein of an amino acid sequence corresponding to thatnucleic acid sequence.

By "expression", "gene expression" or "protein expression" is meant theproduction of protein from information contained within a gene by a hostorganism.

By "transformation" is meant a biochemical method of inducing a hostcell to internalize a nucleic acid molecule. Such nucleic acid moleculesare usually genetic elements comprising at least an origin ofreplication, a selectable marker gene, and a promoter for expression ofthe selectable marker gene within the host cell.

By "heterologous" is meant not of the same species. Thus, an enzymeexpressed in a heterologous host cell is produced in a host cell of adifferent species than the one from which the enzyme was originallyderived.

By "gene" is meant a nucleic acid region having a nucleotide sequencethat encodes an expressible protein or polypeptide. A gene may compriseone or more "coding sequences" containing codons that correspond toamino acid residues of the expressed protein; the gene may alsocomprise, but need not comprise, one or more "non-coding" nucleotidesequence regions that do not contain codons corresponding to amino acidresidues of the expressed protein.

All of the biochemical techniques, used for construction and evaluationof the MMLV-RT expression vectors including, but not limited to,restriction digestion protocols, gel electrophoresis, Southern blot, andDNA modification reactions, are known to those of ordinary skill in theart and are described in Sambrook et al., supra, which was previouslyincorporated by reference herein. In addition, many of these techniquesare described in PCT Publication No. WO 95/27067, entitled "Purified DNAPolymerase from Bacillus stearothermophilus."

I. Construction of the Cloning Vector

a. Plasmid pUC18N (FIG. 1A)

Plasmid pUC18 (Life Technologies, Inc., Bethesda, Md.) was used as theparent vector. Clones were screened by restriction mapping techniques onagarose gels; such techniques are well known in the art. An Nco Irestriction site was introduced between the lac Z ribosome binding siteand the Eco RI restriction site of pUC18 by making a substitution of twonucleotide bases, as shown in FIG. 1B. The mutations were introducedusing the two synthetic oligonucleotides shown in FIG. 2 asoligonucleotides 1 and 2 (SEQ ID NO: 1 and 2 Respectively). As shown,the oligonucleotides overlap by 30 base pairs at their 3' ends. Theoligonucleotides were allowed to hybridize, filled in using the Klenowfragment of E. coli DNA polymerase I, and digested with Pvu II and EcoRI. Plasmid pUC18 was digested with Eco RI and partially digested withPvu II to yield two DNA fragments: a larger fragment including theintact ampicillin resistance gene (Amp), the origin of replication(Ori), and part of the lac Z gene. The smaller Eco RI-Pvu II fragmentconsisted of the portion of the lac Z gene corresponding to positions450 to 628 of the pUC18 map. The synthetic Eco RI-Pvu II fragment wasinserted into the larger vector fragment, ligated and used to transformE. coli strain JM 109. Clones containing properly constructed vectorsproduced a blue color using an X-gal substrate(5-bromo-4-chloro-3-indolyl-β-D-galactoside) as a substrate, indicatingthat the lac Z gene had been properly reconstructed. These results werefurther verified by restriction mapping. This vector was named pUC18N.(See FIG. 1).

b. Construction of Plasmids Containing the Reverse Transcriptase Gene.

The intact MMLV gene was isolated as an Sst I-Hind III fragment from thepMMLV-L clone described in Miller and Verma, J. Virol. 49:214-222(1984). This fragment contained the nucleotide sequence corresponding tothe region from MMLV position 2558 (Sst I site) to position 4894 (HindIII site) and contained the entire RT gene between 40 extra upstreambases and 284 extra downstream bases. Plasmid vector pUC18 was digestedwith Sst I and Hind III, and the vector and RT gene were ligatedtogether and used to transform competent E. coli DH5∝f' cells. Theresulting plasmid was named pUC18 MMLV Sst-Hind (FIG. 5).

This plasmid was then digested with Eco RI and Bgl I, yielding a 2013 bpfragment of the MMLV-RT gene lacking the terminal 3' sequences of the RTgene. The RT gene fragment was ligated at its Bgl I site to adouble-stranded linker designed with Bgl I-Hind III overhangs (FIG. 7)from two synthetic oligonucleotides 8 and 9 (SEQ ID NOS:10 and 11,respectively). The synthetic linker contained the coding sequences forthe carboxyl terminus of MMLV reverse transcriptase and a stop codon.Plasmid pUC18 was digested with Eco RI and Hind III, and the largevector fragment was gel purified and ligated with the reconstructed RTgene. The resulting plasmid was called pUC18 MMLV III Tailed, andcontained the MMLV gene with the extra 3' sequences removed.

c. Constriction of pUC18N MMLV Gly and pUC18N MMLV Gly Tet(-).

The extraneous 5' sequences of the cloned RT gene were removed asfollows. A 1997 bp Mam I-Hind III fragment was isolated from pUC18N MMLVIII Tailed (fragment (3) in FIG. 8A). This nucleic acid fragmentcontained the RT gene without the 5' twenty-three nucleotides of theMMLV-RT gene sequence. Two complementary oligonucleotides weresynthesized and hybridized to recreate the 5' portion of the RT gene(fragment (2) in FIG. 8B) (but with nucleotides coding for a glycine inthe second amino acid position and a Nco I 5' overhang containing aninitiation codon, as shown below.

    Oligonucleotide #3 CATGGGTCTG AACATCGAAG ATGA                                                              (SEQ ID NO:3)                                       - Oligonucleotide #4 TCATCTTCGA TGTTCAGACC (SEQ ID NO:4)                      - 5'-CATGGGTCTGAACATCGAAGATGA-3'                                                 3'-CCAGACTTGTAGCTTCTACT-5'                                          

Plasmid pUC18N was digested with Nco I and Hind III, and the smaller ofthe two resulting fragments was removed. The hybridized oligonucleotides(SEQ ID NO:3 and 4) were ligated to the larger pUC18N fragment (fragment(1) in FIG. 8C) at the Nco I site, and the 1992 bp MMLV-RT Mam I-HindIII gene was then inserted as well, yielding the expression vectorpUC18N MMLV Gly (FIG. 8D). The Tet gene from pUC18 Tet(+) (FIG. 8E),constructed as described below, was inserted at the Aat II site, and theresulting plasmid was called pUC18N MMLV Gly Tet(-) (FIG. 8F). The minussign refers to the orientation of the Tet gene within the vector.

The cloned MMLV-RT of the present invention differs from the nativeenzyme in two respects. First, the codon encoding the threonine residuewhich occupies position 1 of the native enzyme (the second codon of theRT gene) has been replaced with a glycine codon in the cloned RT of thepresent invention; secondly, the codons for the leucine, asparagine andisoleucine residues occupying amino acid positions 2, 3 and 4 of themature native protein sequence were replaced with codons more preferredby E. coli. The CTA codon coding for leucine was replaced withdegenerate codon CTG; the AAT codon coding for asparagine was replacedwith degenerate codon AAC, and the ATA codon coding was replaced withdegenerate codon ATC. (See Wada, K. et al., Nucl. Acids Res.19(supp.):1981-1986 (1991)).

d. Construction of Plasmid pUC18N SD9D

In order to optimize the expression of cloned MMLV-RT, the lac Zribosome binding site (RBS) of pUC18N was modified to contain 9 basescomplementary to E. coli 16S rRNA rather than on the 4 such basespresent in the pUC18 parent vector. At the same time, plasmids wereconstructed having spacer regions separating the RBS and the ATGinitiation codon by either 7, 8, or 9 base pairs, as shown for one ofthe strands in FIG. 3 as SD7, SD8 and SD9. Common elements in the designof these spacer sequences were 1) adenosine (A) in the third position 5'to the ATG initiation codon (i.e., A at -3 position), 2) no guanine (G)or cytosine (C) in the spacer region except in the Nco I site (CCATGG),and 3) an 5'-RRTTTRR-3' sequence spanning the RBS and the spacer, whereT is thymine and R is a purine nucleotide and A or T is between the RBSand ATG. These common elements for heterologous gene expression weresuggested in Jay et al., Proc. Natl. Acad. Sci. USA 78:5543-48 (1981)and Jespers et al., Protein Engineering 4:485-92 (1991).

The oligonucleotides used to introduce these modifications are shown inFIG. 4. Oligonucleotides 5, 6 and 7, respectively (SEQ ID NO:5, 6 and 7)were each used in conjunction with oligonucleotide 1 (SEQ ID NO: 1),shown in FIG. 2. Oligomers 6 and 7 are identical to Oligomer 5 except asindicated in FIG. 4. Oligomers 5, 6 and 7 were used with Oligomer 1 asin the construction of pUC18N. The nucleotide 4 bases on the 5' side ofthe ATG start codon of oligonucleotide 6 and the 4 and 5 bases on the 5'side by the ATG start codon in oligonucleotide 7 were synthesized with amixture of A and T since neither was theoretically preferred. SeeJespers, et al., supra. As in the construction of pUC18N, a 30 base pairregion of complementarity existed between oligonucleotide 1 and each ofoligonucleotides 3, 4 and 5. As before, each pair of oligonucleotideswas allowed to hybridize, was filled in using the Klenow fragment of E.coli DNA polymerase I, digested with Pvu II and Eco RI and inserted intothe same large pUC18 Pvu II-Eco RI fragment used in constructing pUC18N.The MMLV-RT gene was then cloned into this vector as a Nco I-Hind IIIfragment as described below.

These constructs were evaluated by measuring the levels of MMLV-RTexpression. The cells containing the plasmid with the 9-base spacer(SD9; oligonucleotide 7 SEQ ID No: 7) displayed the highest level ofreverse transcriptase expression. The plasmid was isolated andsequenced; both of the degenerate nucleotides 4 and 5 bases on the 5'side of the ATG start codon were found to be adenosine (A) residues. Theexpresssion vector was named pUC18N SD9D.

e. Insertion of the Tetracycline Resistance Gene

The ampicillin resistance (β-lactamase) gene of pUC18 was used as agenetic selection marker in the early vector constructions. However,owing to the fact that β-lactamase acts to destroy the antibioticrelatively quickly, there may be a sizable plasmid-minus revertantpopulation in a culture in which ampicillin is the sole selectivecriterion.

In order to tightly regulate the cell population in the cultures, thevector was modified to contain a tetracycline resistance gene. Becausetetracycline acts to block cellular uptake of the antibiotic rather thaninactivating it, the culture should be more stable in the presence oftetracycline than with ampicillin.

The tetracycline resistance gene was isolated from pBR322 as a 1427 bpEco RI-Ava I fragment. The single strand overhangs were filled in usingthe Klenow fragment of E. coli DNA polymerase I, yielding a blunt-endedfragment. Aat II linkers were ligated to the tetracycline resistancegene fragment, and digested with Aat II. Plasmid pUC18 was digested withAat II, and the linearized vector was ligated to the Aat II fragmentcontaining the tetracycline resistance gene. The ligation mixture wasused to transform competent E. coli JM109 cells, and the transformantswere selected by tetracycline resistance. The structure of the plasmidwas verified by restriction mapping. Clones were selected having thetetracycline resistance gene inserted in both orientations; the plasmidswere named pUC Tet(+) and pUC Tet(-).

The two plasmids were used as a supply of the tetracycline resistancegene (Tet) for insertions into plasmids containing cloned MMLV reversetranscriptase. This approach was preferable to attempting to insert thereverse transcriptase gene(RT) into a vector already containing the Tetgene, since the Tet gene contains restriction sites for enzymes used inthe reverse transcriptase cloning, while the RT gene contains no Aat IIsites.

f. Construction of pUC18N SD9D MMLV Gly and pUC18N SD9D MMLV Gly Tet(-).

The intact, modified reverse transcriptase gene from pUC18N MMLV GlyTet(-) was isolated as a 2018 bp Nco I-Hind III fragment (fragment (2)in FIG. 9A) and ligated with vector pUC18N SD9D from which the NcoI-Hind III polylinker region had been removed (fragment (1) in FIG. 9B).The resulting plasmid, called pUC18N SD9D MMLV Gly (FIG. 9C), containedthe MMLV-RT gene modified in the three ways described above in additionto having the improved ribosome binding site and spacer region, asdescribed above. This plasmid was cleaved at its unique Aat II site, andthe Aat II Tet gene fragment from pUC18 Tet(+) (FIG. 9D) was insertedinto the vector and ligated. Plasmids containing the Tet gene insertwere isolated in both possible orientations, and the level of RTexpression was tested for clones containing each plasmid. The clonehaving the Tet gene in the (-) orientation (with the coding strand inthe same orientation as MMLV-RT); FIG. 9E was found to produce higherlevels of RT than the clone having the Tet gene in the opposingorientation and was therefore chosen as the preferred clone.

II. Selection of the Host Cell Strain

The following E. coli strains were tested for expression andpurification of MMLV-RT: JM109, DH5∝f', XL1 Blue (Stratagene, San Diego,Calif.), JM105, ER 1458, NM 522, Inv∝f' (Invitrogen, San Diego, Calif.),TOPP™ strains 1-6 (Stratagene), 1200, MRE 600, Q 13, and A 19. Some ofthese strains are mutants which are deficient in RNase I (strains 1200,MRE 600, Q 13, and A 19), while others are common laboratory strains.Some of these strains contain the lac I^(q) repressor and requiredinduction with isopropylthiogalactoside (IPTG). The level of RTexpression of host cells containing the RT gene was estimated byvisualization of the resulting protein on SDS-polyacrylamide gels andalso, in most cases, by enzyme activity assays on crude cell lysates. Ofthe RNase I deficient strains, E. coli 1200 (Yale University E. coliGenetic Stock Center, Strain 4449) consistently showed high levels ofenzyme expression using these assays; unless indicated otherwise, allexperiments described herein were conducted using this strain.

III. Growth of E. coli 1200 containing pUC18N SD9D MMLV Gly Tet(-)

The fermentation culture medium (A-Z Amine media) contained thefollowing components in a volume of 200 liters:

    ______________________________________                                        N-Z Amine A (Sheffield Products, Norwich, N.Y.)                                                           2 kg                                                Yeast Extract (Difco)  1 kg                                                   NaCl  1 kg                                                                    NaOH  8 g                                                                     Tetracycline (12 mg/ml in 70% ethanol) 200 ml                               ______________________________________                                    

The mixture was autoclaved in the fermentation vessel at 121° C. for 20minutes, then allowed to cool. The tetracycline was added when thetemperature reached 37° C.

The inoculum of E. coli 1200 containing pUC18N SD9D MMLV Gly Tet(-) wasprepared by inoculating 2 ml of N-Z Amine plus 12 μg/ml tetracycline(LB+Tet) with a frozen stock culture of the vector-containing strain andincubating overnight at 37° C. with shaking. The resulting 2 ml culturewas then used to inoculate 20 one-liter cultures, which were againincubated overnight at 37° C. with shaking.

The 200 liter fermenter was then inoculated with 20 liters of seedculture, and the cells were allowed to grow at 37° C. until 30 minutesafter the culture had reached maximum density as determined by measuringlight attenuation at a wavelength of 660 nm. This generally occurs about7.5 hours after inoculation. During incubation the culture was stirredcontinuously at 150 RPM for the initial 3 hours and then at 180 RPMthereafter. The vessel was sparged with air at 45 l/min. The pH of themedium was not controlled during fermentation, and rose during that timeto approximately 8.2.

The culture was chilled to 20° C., and the cells were collected bycentrifugation in a Sharples centrifuge. The cells were not washed. Thecell paste was divided into 200 g portions and frozen in liquid N₂.During freezing, the cell mass was broken into smaller pieces to ensurerapid and thorough freezing. The frozen cell paste was then stored at-70° C.

IV. Purification of MMLV Reverse Transcriptase from E. coli 1200/pUC18NSD9D MMLV Gly Tet(-)

1. Assay of Reverse Transcriptase Activity and Protein Concentration.

Methods for assaying reverse transcriptase activity are known in theart. For the work described here, the dT:rA assay described by Kacianwas used (Kacian, Methods for Assaying Reverse Transcriptase, in Methodsin Virology (Academic Press 1977), herein incorporated by reference aspart of this disclosure). One unit of reverse transcriptase activityconverts 1 nmole of dTTP to acid-precipitable form in 10 minutes underthe conditions described therein.

2. Cell Lysis

Eleven hundred grams of frozen cell pastes were broken into pieces andsuspended in 3.3 liters of Lysis Buffer (25 mM Tris-HCl (pH 7.5), 10 mMethylenediamine tetraacetic acid (EDTA), 10% (v/v) glycerol, 5 mMdithiolthreitol (DTT), 1% (v/v) Triton X-100, 10 mM NaCl, 1 mMphenylmethylsufonyl fluoride (PMSF)) by stirring at 4° C. The cells werethen lysed by 2 passes through an APV Gaulin 15MR homogenizer at acontinuous pressure of 8,000 psi. The receiving vessel was kept in anice water bath, and the initial homogenate was allowed to chill for 30minutes prior to the second pass. The lysate was then cleared bycentrifugation at 4,500×g for 1 hour at 4° C., and the pellet wasdiscarded. The clarified lysate was either used immediately or storedfrozen at -70° C. and brought to 4° C. before use.

3. Phosphocellulose Column Chromatography

Phosphocellulose (Whatman P11, 100 g) was treated with 2.5 liters of 0.5N NaOH, followed by 2.5 liters of 0.5 N HCl, as recommended by themanufacturer. After a final water wash, the phosphocellulose wassuspended in 1.0 l of 1.0 M Tris-HCl (pH 7.5), allowed to stand for 5-10minutes, and transferred to a Buchner funnel. The buffer was removed byvacuum filtration, and the phosphocellulose was washed with 1.0 MTris-HCl (pH 7.5) until the pH of the effluent matched the pH of thewash solution. The phosphocellulosle was transferred to a beaker andsuspended in 1.0 l of column buffer (25 mM Tris-HCl (pH 7.5), 1 mM EDTA,10% (v/v) glycerol, 1 mM DTT, 0.1% (v/v) Triton X-100 and 1 mM PMSF)containing 0.05 M NaCl. After 5-10 minutes the buffer was removed undervacuum filtration as described above. The phosphocellulose was thensuspended in 700 ml column buffer containing 0.05 M NaCl and cooled to4° C.

All subsequent steps were carried out at 4° C. The chromatography wascarried out using Pharmacia FPLC equipment. A Pharmacia XK 50/30 (5.0cm×26.0 cm) was packed with the washed and equilibrated phosphocelluloseto give a bed of 500 ml. The column was then washed with 1 l of columnbuffer containing 0.05 M NaCl at a flow rate of 60 ml/hour. Columnadapters (Pharmacia AK 50) were used to minimize the dead volume at theends of the column. Six hundred ml of clarified cell lysate were appliedto the column at a flow rate of 30 ml/hour. The column was then washedwith 650 ml of column buffer containing 0.2 M NaCl at the same flowrate. Because of shrinkage of the column bed, excess buffer was removedfrom the space above the column bed, and the top flow adapter wasreadjusted to maintain contact with the bed surface.

The column was eluted with a 1500 ml linear salt gradient, from 0.2 MNaCl to 0.7 M NaCl in a column buffer at 30 ml/hour. The effluent wasmonitored for the presence of protein by its absorbance at 280 nm.Fractions of 25 ml were collected except during the elution of theprotein peak, during which 15 ml fractions were collected.

The column fractions were analyzed using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) followed by Coomassie Brilliant Bluestaining. SDS-PAGE is well known in the art, and is described inLaemmli, U. K., Nature 227:680 (1970), which is hereby incorporated byreference herein. Ten microliters from each fraction were analyzed ineach gel lane (FIG. 10, lanes 3-5). A control lane contained a knownamount of purified MMLV-RT (FIG. 10 lane 2). Those fractions containinga significant amount of protein migrating with an apparent molecularweight similar or identical to that of the MMLV-RT control and whichcontained little visible contaminating protein bands were pooled.Approximately 95% of the protein eluting within the major protein peakwas able to be pooled without including a significant amount ofcontaminating proteins. Activity assays may also be used to locate andpool the peak MMLV-RT enzyme fractions; such procedures are known tothose of ordinary skill in the art.

4. Sephacryl S200 Gel Filtration

The pooled phosphocellulose fractions, having a volume of 80-100 ml,were concentrated to less than 25 ml by ultrafiltration in an Amiconultrafiltration cell using an Amicon P30 membrane at 20 psi of nitrogen.Two 2.6 cm×94 cm Pharmacia XK 26/100 columns were packed with SephacrylS200 (Pharmacia) according to the manufacturer's directions. Columnadapters were used to minimize the dead volume. Both columns wereconnected in series. The columns were washed with 2 l column buffercontaining 0.2 M NaCl at a flow rate of 90 ml/hour. The concentratedphosphocellulose pool (about 25 ml) was loaded onto the upstream column,and the column was developed with the same buffer at a flow rate of 90ml/hour. Again, the effluent was monitored for its absorbance at 280 nm;the initial 200 ml of effluent was collected in a single pool, and 4 mlfractions were collected during the elution of the protein peak. TheMMLV-RT eluted when approximately 290-300 ml of buffer had been appliedto the columns.

The fractions were again analyzed using SDS-PAGE as above. Threemicroliters from each fraction in the peak region were run in each gellane (FIG. 10, lanes 6-10); as before, a control lane contained purifiedMMLV-RT of a known mass. Those fractions containing a significant amountof protein migrating with the purified MMLV-RT and which containedlittle visible contaminating protein were pooled. Preferably, thosefractions containing predominant bands of a higher apparent molecularweight than the MMLV-RT were not included in the pool. Between 95-98% ofthe protein in the major S200 peak was included in the pool. Althoughassays for reverse transcriptase activity may be used to locate andidentify the MMLV-RT in the fractions, analysis preferably includesSDS-PAGE to avoid including higher molecular weight contaminants in thepool.

The pooled S200 fractions are sufficiently concentrated for most uses.The enzyme can be stored in 50% glycerol at -20° C.

EXAMPLE 1 Expression of MMLV-RT by E. coli containing pUC18N MMLV GlyTet(-) or pUC18N MMLV Gly Tet(-) with a Modified Ribosome Binding Siteand Spacer Sequences of Different Lengths

The MMLV-RT gene containing the glycine amino acid substitution in thefirst position was evaluated in vector pUC18N and pUC18N with thespacers and modified ribosome binding site described above. All vectorscontained the Tet gene and were evaluated in E. coli strain 1200.

Fifty ml cultures of E. coli 1200 containing either of these twocontructs were grown for 16.5 hours at 37° C. with shaking. Aliquots of0.5 ml were harvested, centrifuged for 2 minutes in a microcentrifuge,and the supernatants were discarded. The cell pellets were resuspendedon 0.5 ml of a wash buffer (50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 5 mMEDTA and 0.25 M sucrose) and then centrifuged as before. The cellpellets were frozen at -80° C. and then resuspended in 200 μl of lysisbuffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 mM EDTA 1% glycerol, 5 mMDTT, 0.2 mM PMSF and 100 μg/ml lysozyme) and left on ice for 20 minutes.One hundred microliters of 0.75% (v/v) Triton X-100 was added to eachsample, and the mixture was frozen and thawed twice. The lysate wascleared by centrifigation, and total protein was assayed by the methodof Read and Northcote (Anal. Biochem. 116:53-64 (1981)), the disclosureof which is incorporated by reference herein.

Aliquots of the lysate were assayed for reverse transcriptase activity.The level of reverse transcriptase activity in each clone was calculatedin terms of units per microgram of total protein in the lysate, as wellas units per ml of bacterial culture. The results shown in Table 1indicate that the vector containing the modified ribosome binding site(RBS) and the 9 base spacer sequence expressed the highest levels ofenzyme.

                  TABLE 1                                                         ______________________________________                                        Comparison of RT Expression in Different Plasmid Constructs                                      RT activity; U per                                                                        RT activity; U per                               Expression Vector μg total protein ml culture                            ______________________________________                                        unmodified pUC18N                                                                            1.81        746                                                  pUC18N with 7 base 2.25 823                                                   spacer and improved                                                           RBS                                                                           pUC18N with 8 base 1.72 679                                                   spacer and improved                                                           RBS                                                                           pUC18N with 9 base 2.69 1,036                                                 spacer and improved                                                           RBS                                                                         ______________________________________                                    

EXAMPLE 2 Comparision of Modified MMLV-RT in E. coli 1200 and JM 109Host Strains

Plasmid pUC18N was used to create plasmids encoding MMLV-RT withglycine, alanine, or valine substitutions in the first native amino acidposition. These substitutions were created using oligonucleotidessimilar to oligos 3 and 4, but with a codon of sequence 5'-GTT-3' or5'-GCT-3' (coding for valine or alanine respectively) in the secondposition of the RT gene, following the initiation codon. The Tet genefrom pUC18 Tet(+) was inserted into the resulting plasmids in eachorientation for comparison. These plasmids were used to transform E.coli JM109 host cells which contain an episomal copy of the lacrepressor lac I^(q) gene. The transformant cells were grown overnight asin Example 1, except when the cells reached log phase growth, the lacpromoter was induced by the addition of 0.5 mM (IPTG) for approximately22 hours. Aliquots were harvested and assayed for reverse transcriptaseactivity as in Example 1. The results are shown in Table 2 below. As canbe seen, the Gly Tet(-) construction showed the highest level of enzymeexpression.

                  TABLE 2                                                         ______________________________________                                        Effect of Orientation of Tet gene on RT Activity                                         RT Activity; U per                                                                         RT Activity; U per                                      μg Total Protein ml Culture                                              ______________________________________                                        Gly Tet (+)                                                                              0.44         177                                                     Gly Tet (-) 1.29 472                                                          Ala Tet (+) 0.59 229                                                          Ala Tet (-) 0.54 243                                                          Val Tet (+) 0.91 400                                                          Val Tet (-) 1.03 395                                                        ______________________________________                                    

In a separate experiment, the Gly Tet(-) and the Val Tet(-) constructswere evaluated in E. coli hosts 1200 and JM 109. The JM 109 cultureswere induced as above, while the 1200 cultures were uninduced. Theresults shown below in Table 3 indicate that the levels of expression inboth strains are comparable for the Gly substituted MMLV-RT, and higherin strain 1200 for the Val substituted plasmid.

                  TABLE 3                                                         ______________________________________                                        Comparison of RT Expression in Different Host Cell Strains                                 RT Activity; U per                                                                         RT Activity; U per                                    μg Total Protein ml Culture                                              ______________________________________                                        1200/Gly Tet (-)                                                                           1.04         591                                                   JM 109/Gly Tet (-) 1.05 533                                                   1200/Val Tet (-) 1.00 516                                                     JM 109/Val Tet (-) 0.61 357                                                 ______________________________________                                    

EXAMPLE 3 Growth of E. coli 1200/pUC18N SD9D MMLV Gly Tet(-) andExpression of MMLV-RT

One liter of growth medium contained 10 g of N-Z Amine A, 5 g of yeastextract, 5 g of NaCl, and 0.1 ml of 10 N NaOH. One milliliter of 12mg/ml tetracycline in 70% ethanol was added to the cooled, autoclavedmedium.

Two ml of medium was inoculated from a frozen stock culture of the E.coli transformant. This was allowed to grow overnight with shaking at37° C. The two ml bacterial culture was used to inoculate 500 ml ofmedium, and this culture was grown overnight as above. The 500 mlculture was, in turn, used to inoculate 5 liters of medium in a NewBrunswick BioFlo III fermenter. The culture was grown at 37° C. withstirring at 350 RPM. The culture was sparged with air at 4 liters/minuteduring fermentation. Five to ten milliliter samples were taken everyhour for measurement of pH, optical density, protein concentration andreverse transcriptase activity. These results are shown in Table 4below.

                  TABLE 4                                                         ______________________________________                                        Growth Kinetics of 1200/pUC18N SD9D MMLV Gly Tet(-)                                                                       RT                                       RT Activity;                                                                 Protein Activity; U/mg                                                    Sample Time(hr) pH A600 (mg/ml) U/Assay Protein                             ______________________________________                                        1       0*      6.93    0.00 0.00  0.00     0                                   2  0** 7.15 0.29 0.06 1.17 2920                                               3 1 7.14 0.22 0.18 0.91 2270                                                  4 2 6.97 0.69 0.07 0.51 1270                                                  5 3 6.91 0.96 0.11 0.87 2170                                                  6 4 7.02 1.72 0.09 0.92 2300                                                  7 5 7.40 2.16 0.13 0.60 1510                                                  8 6 7.70 2.50 0.11 1.27 3180                                                  9 7 7.83 2.91 0.12 1.49 3720                                                  10 8 7.98 2.79 0.09 1.46 3660                                               ______________________________________                                         *Pre-Inoculation                                                              **PostInoculation                                                        

EXAMPLE 4 Large-Scale Purification of Cloned MMLV-RT

The enzyme was prepared as described in Example 1 above. Volumes ofreagents were adjusted in proportion to the weight of the pelleted cellsat the beginning of the procedure. As indicated in Table 5, highpurified enzyme was recovered with a 48% yield.

                  TABLE 5                                                         ______________________________________                                        Purification Parameters: MMLV-RT Purification Scale-Up                                                   Total   Specific                                      Volume Protein Activity Activity                                             Fraction (ml) (mg) (U) (U/mg) Yield (%)                                     ______________________________________                                        Crude  605.6    2.1 × 10.sup.4                                                                   1.5 × 10.sup.8                                                                   7,100 100                                     Lysate                                                                        P-11 Pool 15.2 741 8.2 × 10.sup.7 110,656 52                            Sephacryl 79 363 7.9 × 10.sup.7 217,400 48                              Pool                                                                        ______________________________________                                    

EXAMPLE 5 SDS-PAGE of Purified MMLV-RT from 1200/pUC18N SD9D MMLV GlyTet(-) Clone

The progress of the purification was monitored by SDS-PAGE analysis ofprotein in the P-11 pool and the Sephacryl pool of Example 4 above.SDS-PAGE was conducted in a 10% reducing gel essentially as described inLaemmli, supra. Samples were prepared as follows. An aliquot of the P-11pool was diluted 50-fold into a gel sample buffer (50 mM Tris-HCl (pH6.8), 10% (v/v) glycerol, 5% β-mercaptoethanol (BME), 2% (w/v) SDS and0.05% (w/v) bromphenol blue) and heated at 95° C. for five minutes. Analiquot from the Sephacryl column pool was diluted 10-fold with gelsample buffer and heated in the same way. A sample of commerciallyobtained MMLV-RT (USB, Cleveland, Ohio) was prepared identically. Thelatter sample was reported by the supplier to have a specific activityof 187,000 U/mg and was provided in an initial concentration of 1500U/μl. Prestained molecular weight markers (Bio Rad Laboratories, SanRafael, Calif.) were used to estimate the molecular weights of theproteins contained in the sample pools. The apparent molecular weight ofthe marker proteins were 18,500 Da (egg white lysozyme), 27,500 Da(soybean trypsin inhibitor), 32,500 Da (bovine carbonic anhydrase),49,500 Da (chicken ovalbumin), 80,000 Da (bovine serum albumin), and106,000 Da (phosphorylase B from rabbit muscle). The gel was loaded asshown in Table 5, and is shown in FIG. 10.

                  TABLE 5                                                         ______________________________________                                        Order of SDS-PAGE Samples in MMLV-RT Purification                               Lane          Sample     Volume (μl)                                     ______________________________________                                        3           P-11 Pool  2.0                                                      4 P-11 Pool 4.6                                                               5 P-11 Pool 7.0                                                               6 P-11 Pool 10.0                                                              7 Sephacryl Pool 6.5                                                          8 Sephacryl Pool 5.0                                                          9 Sephacryl Pool 4.2                                                          10  Sephacryl Pool 3.3                                                      ______________________________________                                    

EXAMPLE 6 Contaminating Ribonuclease Activity in a CommercialPreparation of MMLV-RT

A 24 cm×0.4 cm column of Sephadex G75 was equilibrated with thefollowing buffer (1×Column Buffer) 20 mM Tris-HCL (pH 7.6), 0.1 mM EDTA,200 mM NaCl, 1 mM dithiothrietol (DTT), 0.01% (v/v) Nonidet P-40 and 10%(v/v) glycerol.

RNase assays were performed by using nucleic acid hybridization tomeasure loss of RNA incubated with the enzyme. Details of the method canbe found in Arnold, et al., (U.S. Pat. No. 5,283,174) and in Nelson, etal., (U.S. patent application Ser. No. 08/094,577). Five ml from eachenzyme sample were transferred to a test tube. Ten ml of an in vitrosynthesized RNA transcript (about 1-4 fmol) in water were added, and thereactions were incubated at 37° C. for 1 hour. Fifty ml of an acridiniumester-labelled DNA probe complementary to a region of the RNA transcriptwere added in 0.1 M lithium succinate (pH 4.7), 1.1 M lithium chloride,2% (w/v) lithium lauryl sulphate, 20 mM EDTA, 20 mM ethylene glycol bis(beta-amino ethyl ether) N, N, N¹, N¹ tetraacetic acid (EGTA), 15 mMAldrithiol (Aldrich Chemical Company, Milwaukee, Wis.), and the reactionmixture was incubated at 60° C. for 20 minutes. Three hundred ml of asolution of 0.6 M sodium borate (pH 8.5), 1% (v/v) Triton X-100 wereadded, and the reaction mixture was incubated at 60° C. for 7 minutes todestroy acridium ester present on unlabelled probe. The amount ofremaining label was determined in a luminometer.

Similar methods for assessing RNase activity using radiolabel probes ordirectly measuring degradation of radiolabelled RNA by monitoringconversion from acid-precipitable to acid soluble forms are well knownto those skilled in the art and may be used in the practice of thepresent invention. Other methods for assaying low level RNase activityare available in the scientific literature and their application to thepractice of the present invention is easily appreciated by those ofskill in the art.

Twenty five microliters of a commercial preparation of MMLV-RT (U.S.Biochemicals, Cleveland, Ohio) was mixed with 12.5 μl of 10×ColumnBuffer without glycerol, 10 μl of a 10 mg/ml solution of Blue Dextran,and 77.5 μl water. Before use the water was treated with diethylpyrocarbonate, as described in Sambrook et al., supra, to destroycontaminating RNAses. The enzyme was applied to the column and elutedwith Column Buffer at a flow rate of 1.8 ml/hour. Two hundred thirty μlfractions were collected and assayed for reverse transcriptase and RNAseactivities, as described above.

The results of two identical column runs are shown in the followingtable.

                  TABLE 6                                                         ______________________________________                                        Comparison of Enzyme Activities For Two Different Column Runs                        Column 1         Column 2                                                               RNAse              RNAse                                        RT Activity Activity RT Activity Activity                                    Fraction (RLU) (% degraded) (RLU) (% degraded)                              ______________________________________                                         1                                                                               2   61                                                                        3                                                                             4  1000                                                                       5                                                                             6  2574                                                                       7                                                                             8  1751  1605                                                                 9   20029                                                                    10 14328 14 98216   9                                                         11                                                                            12 143493   0 40619   0                                                       13   43523                                                                    14 21570 51 9299 55                                                           15   26650                                                                    16 11306  0 13490  17                                                         17   1226                                                                     18   4713                                                                     19   1462                                                                     20  2583 64 1379 57                                                           21   1263                                                                     22  913 52 1072  0                                                            23                                                                            24  907 21  44                                                                25                                                                            26  887 56  73                                                                27                                                                            28 21375                                                                      29                                                                            30  8100                                                                    ______________________________________                                    

As this Table illustrates, the commercial enzyme preparation containssignificant endogenous RNAse activity. This RNAse activity is other thanthe RNAse H activity associated with the MMLV-RT enzyme, since itdegrades single-stranded RNA. When analyzed by gel filtrationchromatography, at least four peaks of non-RNAse H RNAse activity areobtained. These peaks may represent four distinct enzymes.Alternatively, they may represent aggregation of one or more protein,dissociation of such a protein into subunits, or other chromatographicartifacts. At least one of these peaks of non-RNAse H RNAse activityco-elutes with the MMLV-RT.

EXAMPLE 7 Comparison of Contaminating Ribonucleases in PartiallyPurified Recombinant MMLV-RT from E. Coli Host Cells JM 109 and 1200

In order the compare the amount of contaminating ribonuclease activitiespresent in MMLV-RT-containing cell lysates after P11 column purificationbetween host cells JM 109 and 1200 transformed with plasmid pUC18N SD9DMMLV Gly Tet(-), fractions from each column were assayed for reversetranscriptase activity using the dT:rA assay described in Kacian, Meth.Virol. supra, and for non-RNAse H RNAse activity using the assaydescribed in the previous example.

The results obtained for each cell type are shown in the followingtables:

                  TABLE 7                                                         ______________________________________                                        E. Coli Strain 1200                                                                            RT Activity                                                                             RNAse activity                                       Fraction (RLU) (% degraded)                                                 ______________________________________                                         1            1007     0                                                         5  1084 0                                                                    10  1021 0                                                                    15  3712 0                                                                    20  38359 0                                                                   25  20741 0                                                                   30 316513 0                                                                   33 346922 0                                                                   36 504196 0                                                                   39 387533 0                                                                   42 371897 0                                                                   45 472248 0                                                                   48 1199993  0                                                                 51 1529015  0                                                                 54 1126592  0                                                                 57 1034428  0                                                                 60 850009 0                                                                   63 698462 0                                                                   66 390121 0                                                                   69 177736 0                                                                   72 260049 0                                                                   76                                                                          ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        E. Coli Strain JM 109                                                                          RT Activity                                                                             RNAse activity                                       Fraction (RLU) (% degraded)                                                 ______________________________________                                         1          1103        0                                                        5  1238  0                                                                   10  1287  0                                                                   15  28359 29                                                                  20  50927 75                                                                  25  29551 70                                                                  30 350732 83                                                                  35 198151 30                                                                  38 164047 54                                                                  41 149647 66                                                                  44 161963 62                                                                  47 674123 81                                                                  50 2060603  83                                                                53 2703286  85                                                                56 1967435                                                                    59 1608490  90                                                                62 782936 86                                                                  65 265569 78                                                                  68 147948 63                                                                  71  78481 38                                                                  74  44426  3                                                                  77  19964  0                                                                  81  13900  0                                                                ______________________________________                                    

The data show that the enzyme prepared from JM 109 cells containedsignificant amounts of non-RNAse H ribonuclease activity throughout theP11 column profile. Significant amounts of RNAse activity eluted withthe reverse transcriptase activity. In contrast, the reversetranscriptase purified from the E. coli 1200 cells was free ofdetectable contaminating RNAse activity after the crude extract waspurified by phosphocellulose column chromatography.

EXAMPLE 8 Amplification of Mycobacterium tuberculosis Ribosomal RNATarget Sequence Using Purified Recombinant MMLV Reverse Transcriptasefrom E. coli 1200/pUC18N SD9D MMLV Gly Tet(-)

Nucleic acid amplification was performed using the procedure describedin Kacian and Fultz, EPO 0 408 295 A2, which is incorporated byreference herein and which enjoys common ownership with the presentapplication. A reagent mixture was made up as follows: 768 microlitersof water was given, in order, 25 μl 1 M Tris-HCl (pH 8.0), 50 μl 1 MMgCl₂, 44 μl KCl, 500 μl 40 mM rNTPs, 500 μl 10 mM dNTPs, 9 μl T7promoter-primer (84 pmoles/μl), and 5 μl non-T7 primer (150 pmole/μl)and mixed. The volume of this mixture (Solution A) was calculated to besuitable for 50 assays. Forty μl of solution A was added to eachreaction tube. Ten microliters of the purified target rRNA (0.05-25 fgμldiluted in Template Dilution Buffer (0.2% (w/v) bovine serum albumin in150 mM NaCl)) was added to each tube. The target rRNA had nucleic acidsequences sufficiently complementary to the primer and thepromoter-primer to allow hybridization to occur under stringenthybridization conditions. Preporation of rRNA is known to those of skillin the art. Two hundred microliters of silicone oil was layered onto thesurface of each reaction mixture, and the reaction tubes were heated at95° C. for 15 minutes in a heating block. The reaction tubes were thentransferred into a 42° C. water bath and allowed to cool for 5 minutes.

An enzyme mixture was prepared by transferring 46.8 μl Dilution Bufferto a tube and adding 1.1 μl (900 U) MMLV-RT and 2 μl (400 U) T7 RNApolymerase. This mixture was then added to each tube. The reactions werethen incubated at 42° C. for two hours.

The amount of amplified RNA generated was then measured using anacridinium ester-labeled DNA probe directed to the target sequence asdescribed in Arnold et al., PCT WO89/02476 and Arnold et al., Clin.Chem. 35:1588-1594 (1989) the former of which enjoys common ownershipwith the present invention, and both of which are incorporated byreference herein. All reactions were run in quadruplicate except for thenegative control, which was run in duplicate. The results shown in Table9 below indicate that saturating levels of the amplified target sequenceare obtained with as little as 2.5 fg of input template RNA at thebeginning of the experiment.

                  TABLE 9                                                         ______________________________________                                        Sensitivity of Enzyme Preparation in an Amplification Reaction                     Amount of Template RNA Added                                               (fg) Signal (RLU)                                                           ______________________________________                                        250                 2841164                                                      2802308                                                                       2828732                                                                       2828837                                                                      25 2801357                                                                     2968585                                                                       2748909                                                                       2723562                                                                      2.5 2761901                                                                    2809799                                                                       2932942                                                                       2906826                                                                      0   2246                                                                         2443                                                                     ______________________________________                                    

EXAMPLE 9 Synthesis of cDNA Using Purified Recombinant MMLV-RT from E.coli 1200/pUC18N SD9D MMLV Gly Tet(-)

The ability of the recombinant purified MMLV-RT to synthesize cDNA wascompared to that of a commercially available reverse transcriptasepreparation (U.S. Biochemicals) in an RNA sequencing reaction.

TTE buffer was prepared by mixing 20 ml 1 M Tris-HCl (pH 7.5), 0.4 mMEDTA (pH 8.0) and 281.7 μl triethylamine. Primers had the followingsequences:

    5'-TACCTTGTTACGACTTCACCCCA-3'                                                                       SEQ ID NO:8                                                - 5'-CTTAGATGCTTTCAGC-3' SEQ ID NO:9                                   

The primer were labeled with ³² P at their 5' ends using polynucleotidekinase; procedures for end-labelling nucleic acids are generally knownin the art. After being end labelled, the primers were purified bychromatography on Nensorb columns (New England Nuclear) according to themanufacturer's specifications, followed by ethanol precipitation.

Reactions were carried out using either purified recombinant MMLV-RTfrom E. coli 1200/pUC18N SD9D MMLV Gly Tet(-) or reverse transcriptasepurchased from a commercial vendor.

Reaction mixtures contained the following reagents in 100 μl finalvolume: Ten microliters of GPE Buffer (500 mM Tris-HCl (pH 7.6), 175 mMMgCl₂, 250 mM KCl, 20 mM spermidine), 8 μof stock rNTPs (25 mM rCTP andrUTP; 65 mM rATP and rGTP), 4 μl of stock dXTPs (10 mM), 0.5 μl 1 M DTT,20 pmoles ³² P-labelled primer, 20 pmole unlabelled primer, 20 pmolespurified E. coli rRNA, 600 U reverse transcriptase. Reactions wereestablished by mixing all components without the reverse transcriptase,then heating the mixture at 95° C. for 5 minutes to denature thetemplate RNA secondary structure. Reactions were then placed at 60° C.for 30 minutes to allow the primers to anneal to the rRNA target. Thereaction mixture was cooled to room temperature, and the reversetranscriptase was added. DNA synthesis was carried out at 42° C. for 60minutes. Reactions were analyzed on 7% polyacrylamide gels essentiallyas described in Williams et al., BioTechniques 4:138-147 (1986).

Both enzymes were found to synthesize cDNA from the RNA template withequal efficiency as judged from the gel electrophoretograms.

EXAMPLE 10 Reverse Transcriptase-Mediated PCR Using Recombinant MMLV-RTfrom E. coli 1200/pUC18N SD9D MMLV Gly Tet(-)

All PCR reactions were run in a Perkin Elmer-Cetus Model 9600 DNAthermal cycler. The thermal cycler was programmed to incubate thereaction in the following manner and sequence:

94° C. for 3 minutes;

35 cycles between 51° C. for 30 seconds, 72° C. for 2 minutes, and 94°C. for 1 minute;

72° C. for 5 minutes;

4° C. overnight.

Two separate preparations of MMLV-RT were used for this experiment, aswell as a lot from the same commercial vendor as above. Differentamounts of RT were tested, but 50 U of the enzyme was found to beoptimum for all enzyme preparations used. The reagents used in theexperiment were as follows: 5×RT Buffer (50 mM Tris HCl (pH 8.3), 75 mMKCl, 3 mM MgCl₂, 5 mM DTT); 10×PCR Buffer (Perkin Elmer)(100 mM Tris-HCl(pH 8.3), 500 mM KCl, 15 mM MgCl₂, 0.1% gelatin); RT Premix (for eachreaction) (4 μl 5×RT Buffer, 0.8 μl of a 25 mM solution of each dNTP, 50units RT, 100 moles (-) sense primer, water in a total volume of 20 μ);PCR Premix (for each reaction) (8 μl 10×PCR Buffer, 100 pmoles (+) senseprimer, 2.5 units Taq DNA Polymerase, and water to a total volume of 80μl . Probes were stored in 10 mM lithium succinate buffer (pH 5.0), 0.1%lithium lauryl sulfate (LLS).

The probes and primers used for this experiment were designed to becomplementary to sequences of the human papilloma virus (HPV) genome.The probes were labeled with acridinium ester as disclosed in Arnold andNelson, PCT Patent Application No. WO89/02476, hereby incorporated byreference herein.

Crude preparations of unspliced template RNA were made by suspendingSiHa cells (which contain HPV nucleic acid sequences integrated intotheir genome) at a concentration of 1.6×10⁷ cells/ml in 10 mM sodiumphosphate (pH 7.6), 100 mM NaCl. The cells were heated for 15 minutes at95° C., cooled to room temperature, then diluted into water to thedesired concentration. RNA transcripts from the E6 gene were prepared byin vitro transcription of DNA from a plasmid containing the HPV16 E6gene. This plasmid was constructed by cloning a DNA fragment from theHPV clone described by Matsukura et al., J. Virol. 58:979-982 (1986)into pBluescript™ II SK (+) and (-) sense cloning vectors. (Stratagene,San Diego, Calif.) These references are incorporated by referenceherein. RNA transcripts were prepared as indicated by the manufacturer.

The amplification reactions were conducted as follows. Target nucleicacids were added to the MMLV-RT premix. This mixture was heated at 95°C. for 2 minutes. The primers were added and allowed to anneal to thetarget nucleic acids for 10 minutes at 60° C. The reaction mixture wasthen cooled on ice. Reverse transcriptase was added, and the reactionwas incubated at 37° C. for 30 minutes. The reaction was then heated at95° C. for 10 minutes to inactivate the reverse transcriptase. Themixture was cooled in ice, and two drops of mineral oil were layeredonto the surface of each tube.

Taq DNA polymerase, was diluted into the PCR Premix at the concentrationindicated above. Eighty microliters of the PCR Premix was then added toeach sample. The samples were placed in the thermal cycler at 95° C.,and cycling was performed as described above.

Hybridization and detection were carried out as described in Arnold andNelson, supra. For each hybridization assay, 30 μl of water was given 10μl of a PCR reaction mixture. The DNA was denatured at 95° C. for 5minutes. Ten microliters of diluted probe was added and mixed. The tubeswere then incubated at 60° C. for 15 minutes. Three hundred microlitersof selection reagent was added, the tubes were mixed and incubated at60° C. for 5 minutes. The tubes were then cooled in ice, and theremaining acridinium ester label was measured in a LEADER luminometer(Gen-Probe Incorporated, San Diego, Calif.).

The results are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                                            Origin of                                                   Copies of  Reverse Average Net                                                Template RNA RNA Type Transcriptase RLU                                     ______________________________________                                        1 × 10.sup.7                                                                     SiHa cell  commercial    423,084                                        lysate                                                                       1 × 10.sup.7 SiHa cell E. coli 445,003                                   lysate 1200/pUC18N                                                             SD9D MMLV Gly                                                                 Tet (-)                                                                     1 × 10.sup.7 E6 transcript commercial 2,741,628                         1 × 10.sup.7 E6 transcript E. coli 2,291,786                              1200/pUC18N                                                                   SD9D MMLV Gly                                                                 Tet (-)                                                                     1 × 10.sup.4 E6 transcript commercial 103,501                           1 × 10.sup.4 E6 transcript E. coli 1,395,572                              1200/pUC18N                                                                   SD9D MMLV Gly                                                                 Tet (-)                                                                     1 × 10.sup.5 E6 transcript commercial 1,317,386                         1 × 10.sup.5 E6 transcript E. coli 2,283,979                              1200/pUC18N                                                                   SD9D MMLV Gly                                                                 Tet (-)                                                                     1 × 10.sup.6 E6 transcript commercial 1,661,390                         1 × 10.sup.6 E6 transcript E. coli 2,951,045                              1200/pUC18N                                                                   SD9D MMLV Gly                                                                 Tet (-)                                                                     1 × 10.sup.7 E6 transcript commercial 2,294,856                         1 × 10.sup.7 E6 transcript E. coli 2,421,754                              1200/pUC18N                                                                   SD9D MMLV Gly                                                                 Tet (-)                                                                   ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:    13                                       - -  - - (2) INFORMATION FOR SEQ ID NO:    1:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         114                                               (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - GGCATGCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC  - #                  50                                                                        - - GCAACGCAAT TAATGTGAGT TAGCTCACTC ATTAGGCACC CCAGGCTTTA  - #                 100                                                                         - - CACTTTATGC TTCC              - #                  - #                      - #    114                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    2:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         112                                               (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - AAGCTCGAAT TCGTAATCAT GGCCATGGCT GTTTCCTGTG TGAAAGTTTT  - #                  50                                                                         - - ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA  - #                 100                                                                         - - GCCTGGGGTG CC              - #                  - #                      - #      112                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    3:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         24                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CATGGGTCTG AACATCGAAG ATGA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:    4:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         20                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - TCATCTTCGA TGTTCAGACC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    5:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         115                                               (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - GAGCTCGAAT TCGTAATCAT GGCCATGGTT TAAACCTCCT TAGTGAAATT  - #                  50                                                                         - - GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT  - #                 100                                                                         - - AAAGCCTGGG GTGCC              - #                  - #                      - #   115                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    6:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         116                                               (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - GAGCTCGAAT TCGTAATCAT GGCCATGGTW TTAAACCTCC TTAGTGAAAT  - #                  50                                                                         - - TGTTATCCGC TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG  - #                 100                                                                         - - TAAAGCCTGG GGTGCC             - #                  - #                      - #   116                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    7:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         117                                               (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - GAGCTCGAAT TCGTAATCAT GGCCATGGTW WTTAAACCTC CTTAGTGAAA  - #                  50                                                                         - - TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA AGCATAAAGT  - #                 100                                                                         - - GTAAAGCCTG GGGTGCC             - #                  - #                      - #  117                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    8:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         23                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - TACCTTGTTA CGACTTCACC CCA           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:    9:                                 - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         16                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - CTTAGATGCT TTCAGC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:    10:                                - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         46                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - AGGCAGCCAT CACAGAGACT CCAGACACCT CTACCCTCCT CTAATA   - #                     46                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         53                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - AGCTTATTAG AGGAGGGTAG AGGTGTCTGG AGTCTCTGTG ATGGCTGCCT  - #                  50                                                                         - - TTC                  - #                  - #                  - #                 53                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         15                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - ATGGGTCTGA ACATC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:     - #         19                                                (B) TYPE:     - #           nucleic acid                                      (C) STRANDEDNESS:   - #     single                                            (D) TOPOLOGY:    - #        linear                                   - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - TAAGGAGGTT TAAAAAACC             - #                  - #                      - # 19                                                                 __________________________________________________________________________

What is claimed is:
 1. A composition comprising an Escherichia coli hostcell containing a recombinant expression vector, wherein saidrecombinant expression vector comprises a gene derived from a Moloneymurine leukemia virus (MMLV) sequence, said gene encoding an enzymecomprising a DNA-dependent DNA polymerase activity and an RNA-dependentDNA polymerase activity, wherein said gene expresses said enzyme withinsaid host cell, and wherein said host cell has about 0.1% or less ofwild type RNase I activity.
 2. A composition comprising an Escherichiacoli cell having about 0.1% or less of wild type RNase I activitycontaining:i) a recombinant expression vector which comprises a geneencoding a mature native-length Moloney murine leukemia virus (MMLV)reverse transcriptase, and ii) reverse transcriptase enzyme expressed bysaid recombinant expression vector.
 3. A composition comprising:anEscherichia coli host cell that has about 0.1% or less of wild typeRNase I activity; a recombinant expression vector contained within saidhost cell, wherein said vector comprises a gene derived from a Moloneymurine leukemia virus (MMLV) sequence, said gene encoding an enzyme,said enzyme comprisinga DNA-dependent DNA polymerase activity, anRNA-dependent DNA polymerase activity, and an RNAse H activity,andwherein said vector expresses said gene encoding said enzyme within saidhost cell.
 4. The composition of claim 3 wherein said vector furthercomprises a ribosome binding site located 5' to said gene and aninitiation codon located at the 5' boundary of said gene's codingregion.
 5. The composition of claim 4 wherein said vector furthercomprises a nucleotide base sequence consisting of the sequence RRTTTRRlocated 5' to said gene's coding region.
 6. The composition of claim 4wherein from 7 to 9 nucleotide bases separate said ribosome binding siteand said initiation codon.
 7. The composition of claim 6 wherein 9nucleotide bases consisting of the sequence TTAAAAACC separate saidribosome binding site and said initiation codon.
 8. The composition ofclaim 4 wherein said initiation codon is wholly or partly containedwithin a restriction endonuclease recognition sequence.
 9. Thecomposition of claim 8 wherein said restriction endonuclease recognitionsequence is recognized by restriction endonuclease Nco I.
 10. Thecomposition of claim 6 wherein the gene encoding said enzyme includesthe sequence consisting of CTGAACATC coding for amino acids 2 to 4 ofsaid enzyme from the enzyme's amino terminus.
 11. The composition ofclaim 8 wherein the gene encoding said enzyme includes the sequenceconsisting of CTGAACATC coding for amino acids 2 to 4 of said enzymefrom the enzyme's amino terminus.
 12. The composition of claim 1 whereinsaid enzyme encoded by said gene further comprises an RNAse H activity.13. The composition of claim 2 wherein said reverse transcriptase enzymecomprises an RNAse H activity.
 14. The composition of claim 1 whereinsaid enzyme encoded by said gene consists of the same number of aminoacids as mature native viral Moloney murine leukemia virus (MMLV)reverse transcriptase.
 15. The composition of claim 1 wherein said hostcell is Escherichia coli strain 1200, strain MRE 600, strain Q 13 orstrain A
 19. 16. The composition of claim 15 wherein said host cell isEscherichia coli strain
 1200. 17. The composition of claim 3 whereinsaid host cell is Escherichia coli strain 1200, strain MRE 600, strain Q13 or strain A
 19. 18. The composition of claim 17 wherein said hostcell is Escherichia coli strain
 1200. 19. The composition of claim 11wherein said host cell is Escherichia coli strain 1200, strain MRE 600,strain Q 13 or strain A
 19. 20. The composition of claim 19 wherein saidhost cell is Escherichia coli strain
 1200. 21. The composition of claim2 wherein said cell is Escherichia coli strain 1200, strain MRE 600,strain Q 13 or strain A
 19. 22. The composition of claim 21 wherein saidcell is Escherichia coli strain 1200.